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An Analysis of the Ramosa1 Pathway in Zea Mays Utilizing CRISPR/Cas9 Knockouts

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An Analysis of the Ramosa1 Pathway in Zea Mays Utilizing CRISPR/Cas9 Knockouts Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2019 An analysis of the ramosa1 pathway in Zea mays utilizing CRISPR/Cas9 knockouts Ryan James Arndorfer Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Agriculture Commons, Genetics Commons, and the Plant Sciences Commons Recommended Citation Arndorfer, Ryan James, "An analysis of the ramosa1 pathway in Zea mays utilizing CRISPR/Cas9 knockouts" (2019). Graduate Theses and Dissertations. 17391. https://lib.dr.iastate.edu/etd/17391 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. An analysis of the ramosa1 pathway in Zea mays utilizing CRISPR/Cas9 knockouts by Ryan Arndorfer A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Genetics and Genomics Program of Study Committee: Erik Vollbrecht, Major Professor Shuizhang Fei Philip Becraft The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this thesis. The Graduate College will ensure this thesis is globally accessible and will not permit alterations after a degree is conferred. Iowa State University Ames, Iowa 2019 Copyright © Ryan Arndorfer, 2019. All rights reserved. ii DEDICATION Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum. "On the other hand, we denounce with righteous indignation and dislike men who are so beguiled and demoralized by the charms of pleasure of the moment, so blinded by desire, that they cannot foresee the pain and trouble that are bound to ensue; and equal blame belongs to those who fail in their duty through weakness of will, which is the same as saying through shrinking from toil and pain. These cases are perfectly simple and easy to distinguish. In a free hour, when our power of choice is untrammelled and when nothing prevents our being able to do what we like best, every pleasure is to be welcomed and every pain avoided. But in certain circumstances and owing to the claims of duty or the obligations of business it will frequently occur that pleasures have to be repudiated and annoyances accepted. The wise man therefore always holds in these matters to this principle of selection: he rejects pleasures to secure other greater pleasures, or else he endures pains to avoid worse pains." Cicero, Marcus Tullius, and H. (Harris) Rackham. De finibus bonorum et malorum. London, W. Heinemann; New York, The Macmillan Co., 1914. Internet Archive, http://archive.org/details/definibusbonoru02cicegoog. iii TABLE OF CONTENTS Page LIST OF FIGURES .........................................................................................................................v LIST OF TABLES ........................................................................................................................ vii ACKNOWLEDGMENTS ........................................................................................................... viii ABSTRACT ................................................................................................................................... ix CHAPTER 1. GENERAL INTRODUCTION ................................................................................1 History of CRISPR .................................................................................................................... 3 CRISPR Use in Plants ............................................................................................................... 6 References ................................................................................................................................. 8 Figures ..................................................................................................................................... 12 CHAPTER 2. CRISPR/CAS9 KNOCKOUT EXPERIMENT OF PUTATIVE RAMOSA1 INTERACTORS ............................................................................................................................14 Abstract .................................................................................................................................... 14 Introduction ............................................................................................................................. 14 CRISPR/Cas9 system ......................................................................................................... 18 Materials and Methods ............................................................................................................ 23 Choosing Genes for Cas9-mediated knockout ................................................................... 23 Selecting CRISPR Guides .................................................................................................. 26 Guide location selection ..................................................................................................... 27 Description and Assembly of the Cas9 Expression System ............................................... 27 Array screening .................................................................................................................. 29 gRNA cassette assembly .................................................................................................... 30 Synthetic 8x array ............................................................................................................... 30 Transformation into Agrobacterium tumefaciens EHA101 ............................................... 31 Array screening in Agrobacterium tumefaciens ................................................................. 32 Agrobacterium transformation of Maize Hi-II ................................................................... 34 Results ..................................................................................................................................... 35 Sequence analysis uncovers novel mutations in all target genes with varied efficiency ... 36 Discussion ................................................................................................................................ 37 ramosa1 mutations create interesting future study possibilities ........................................ 37 jmjC guides produced mostly frame-shift early termination mutations ............................. 39 selT guides were both highly active ................................................................................... 39 ail6 guides were largely inactive ........................................................................................ 40 rho-GDI1 mutagenesis was limited by ear availability ...................................................... 41 Multi-guide strategy is prudent and functional .................................................................. 41 References ............................................................................................................................... 43 Figures ..................................................................................................................................... 46 Tables ....................................................................................................................................... 72 iv GENERAL CONCLUSIONS ........................................................................................................92 APPENDIX. YEAST-2-HYBRID GENE CANDIDATE INFORMATION ................................93 v LIST OF FIGURES Page Figure 1-1: Meganucleases, ZFN and TALENs diagrams............................................................ 12 Figure 1-2: Bacterial CRISPR array immune system diagram ..................................................... 13 Figure 2-1: Original ramosa ear discovered by Dr. Walter Gernert. ............................................ 46 Figure 2-2: Zinc finger and ramosa1 folding models. .................................................................. 47 Figure 2-3: ramosa1 zinc finger recognition sequence logo. ....................................................... 48 Figure 2-4: ramosa1 phenotype in tassel and ear. ........................................................................ 49 Figure 2-5: Yeast-2-Hybrid description. ....................................................................................... 50 Figure 2-6: CRISPR/Cas9 function. ............................................................................................. 51 Figure 2-7: Screen-shot of University of Bergen’s chop chop V2 program depicting potential guides for ramosa1. ................................................................................... 52 Figure 2-8: ramosa1 gene model. ................................................................................................. 52 Figure
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